Live cell imaging. Why live cell imaging? Live cell analysis provides direct spatial and temporal information Planning your experiment – The markers/fluorophores.

Slides:



Advertisements
Similar presentations
Part 1: The root of all evil Part 2: Fluorescence microscopy
Advertisements

Fluorescence and Confocal Microscopy
Victor Sourjik ZMBH, University of Heidelberg
Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins David Stepensky.
Big Question: We can see rafts in Model Membranes (GUVs or Supported Lipid Bilayers, LM), but how to study in cells? Do rafts really exist in cells? Are.
Live-Cell Imaging of Focal Adhesions Peyton Lab Journal Review Dannielle Ryman May 1, 2012.
ABRF meeting 09 Light Microscopy Research Group. Why are there no standards? Imaging was largely an ultrastructure tool Digital imaging only common in.
 TIRF, FRAP, photoactivation
Fluorophores bound to the specimen surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected.
Today’s Topic: Lec 3 Prep for Labs 1 & 2 3-D imaging—how to get a nice 2D Image when your samples are 3D. (Deconvolution, with point scanning or with Wide-field.
Fluorescence microscopy – Principle and practical consideration Hiro Ohkura.
Chemical Structure of the Chromophore Biosynthesis of the Chromophore Critcial dehyrogenation reaction to juxtapose aromatic group with imidazlinone.
Non-radiative energy transfer from excited species to other molecules
Immunolabeling & Fluorescent Microscopy Presented by: Sumble Maha Khan ABE Workshop June 13 – 30, 2006.
Microscopy is about a combination of resolution (seeing smaller and smaller things), and contrast (seeing what you want to see). Both aspects have recently.
Methods: Single-Molecule Techniques Biochemistry 4000 Dr. Ute Kothe.
Microscopy is about a combination of resolution (seeing smaller and smaller things), and contrast (seeing what you want to see). Both aspects have recently.
Study of Protein Association by Fluorescence-based Methods Kristin Michalski UWM RET Intern In association with Professor Vali Raicu.
Fluorescence Microscopy Chelsea Aitken Peter Aspinall.
Methods, Part 2 February 9, Learning Outcomes Discriminate between different types of microscopy, and justify their use for answering research questions.
Practical aspects of fluorescence microscopy
FRET and Other Energy Transfers Patrick Bender. Presentation Overview Concepts of Fluorescence FRAP Fluorescence Quenching FRET Phosphorescence.
Are You FRETting? Find Out for Sure With FLIM Frequency Domain FLIM for Your Scope Intelligent Imaging Innovations.
FRET(Fluorescent Resonance Energy Transfer)
TIRF Total Internal Reflection Fluorescence Microscopy specialized fluorescence microscopy technique specifically images a very thin optical section (50-250nm)
Fluorescence Techniques
Single molecule pull-down Jain et al, Nature 473:484 (2011) Main points to cover fluorescence TIRF microscopy main advantage evanescent field depth single-fluor.
Lecture 7: Fluorescence: Polarization and FRET Bioc 5085 March 31, 2014.
The current state of Confocal Scanning Laser Microscopy Hjalmar Brismar Cell Physics, KTH.
Powerpoint Templates Page 1 Powerpoint Templates Spectroscopic Microscopy.
Functional cellular imaging by light microscopy MICROSCOPIES.
Advanced Fluorescence & Confocal Microscopy 08/2007 Lecture by Dr. Dirk Lang Dept. of Human Biology UCT Medical School Room Phone:
FAT Average lifetime (ps) GFP- Pax GFP-Pax + FAT- mCherry Lifetime (ns) Pax FAT Advanced Fluorescence Microscopy I: Fluorescence (Foster)
Confocal Laser Scanning Microscopy: general considerations and techniques Simone Bossi.
Today’s take-home lessons (i.e. what you should be able to answer at end of lecture) FRET – why it’s useful, R -6 dependence; R 0 (3-7 nm), very convenient.
The Plasmamembrane and Lipid Rafts 08/2007 Lecture by Dr. Dirk Lang Dept. of Human Biology UCT Medical School Room Phone:
Förster Resonance Energy Transfer (Chemistry/Biology Interface) Michelle, Pauline, Brad, Thane, Hill, Ming Lee, Huiwang Facilitator: Nancy.
Today’s Announcements 1.Next Tuesday: Diffusion (Why moving in a cell is like swimming in concrete.) 2. Homework assigned today Last graded Homework:
FRET 발표자 최예림.
Today’s Topic (02/02/15) How did 1st week of labs go?
IPC Friedrich-Schiller-Universität Jena 1 Radiationless excitation energy transfer requires interaction between donor and acceptor  Emission spectrum.
(Image: T. Wittman, Scripps) Principles & Practice of Light Microscopy 6 Special Techniques (FRET, FRAP, FLIP, FLIM, FCS, molecular sensors…)
Designing a Microscopy Experiment Kurt Thorn, PhD Director, Image from Susanne Rafelski, Marshall lab.
Imaging.
FRET and Biosensors Kurt Thorn Nikon Imaging Center Image: Thomas Huckaba.
Date of download: 7/7/2016 Copyright © 2016 SPIE. All rights reserved. The spectral overlap of Cerulean or mTFP with Venus is compared. The excitation.
Physics 598BP Today: Fluorescence What is it? Why is it good?
Figure 5. A model for rolling-circle amplification of telomeres
Measuring Fluorescence Resonance Energy Transfer in vivo
Jennifer Colby Research Advisor: Dr. Chris Janetopoulos
Reliable and Global Measurement of Fluorescence Resonance Energy Transfer Using Fluorescence Microscopes  Zongping Xia, Yuechueng Liu  Biophysical Journal 
Today’s take-home lessons: FRET (i. e
New Turf for CFP/YFP FRET Imaging of Membrane Signaling Molecules
Fluorescence Applications in Molecular Neurobiology
Today’s take-home lessons: FRET (i. e
Quantification of Membrane Protein Dynamics and Interactions in Plant Cells by Fluorescence Correlation Spectroscopy  Xiaojuan Li, Jingjing Xing, Zongbo.
Volume 83, Issue 6, Pages (December 2002)
Förster Resonance Energy Transfer (FRET)
Research Techniques Made Simple: Methodology and Applications of Förster Resonance Energy Transfer (FRET) Microscopy  Joshua A. Broussard, Kathleen J.
New Turf for CFP/YFP FRET Imaging of Membrane Signaling Molecules
Single-Molecule Microscopy Reveals Plasma Membrane Microdomains Created by Protein-Protein Networks that Exclude or Trap Signaling Molecules in T Cells 
Volume 23, Issue 3, Pages (February 2013)
Asako Sawano, Hiroshi Hama, Naoaki Saito, Atsushi Miyawaki 
FLUORESCENCE MICROSCOPY
Spatially Selective Two-Photon Induction of Oxidative Damage
Volume 31, Issue 6, Pages (September 2001)
Protein Kinase D Inhibitors Uncouple Phosphorylation from Activity by Promoting Agonist-Dependent Activation Loop Phosphorylation  Maya T. Kunkel, Alexandra C.
Imaging techniques for next generation plant cell biology.
Polarized Fluorescence Resonance Energy Transfer Microscopy
Volume 17, Issue 7, Pages (July 2010)
Presentation transcript:

Live cell imaging

Why live cell imaging? Live cell analysis provides direct spatial and temporal information Planning your experiment – The markers/fluorophores – The cell’s environment – Practical aspects of the experiment: the microscope – Photodamage Applications of live cell imaging

Select your markers carefully You only see a limited number of molecules/fluorophores 2 to3 channels in live cell imaging

Fluorophores Usually tag: GFP, mCherry, Venus, dTomato, etc… Transient transfections Overexpression Inducible expression Endogenous levels of plasmid at endogenous promoter

What you need to do Keep the cells happy Optimize your experiment to get the most out of it Limit photodamage (cells will change their behavior)

Key components Preparation and holding of the cell specimen Temperature and CO 2 control Microscope Light: wavelength, intensity Image acquisition Type of live-cell imaging experiments

Unhappy cells

Contamination in cells will affect your experiment And Mycoplasma!

Media types in human cells Need FBS DMEM/RPMI: culture media, contains phenol red, which causes background fluorescence! CO 2 -independent media –for long experiments Leibowitz L15 media, no phenol red!

Holders Must have a #1.5 coverslip (0.17mm thick)

Maintaining live cells on the microscope Tight control of the environment is critical for successful live-cell imaging Heat within the specimen chamber or chamber holder Warm air stream over the stage Enclose the stage area/whole microscope Use CO 2 -independent media Use CO 2 source

Heated objectives Alternatively, need to heat the chamber and lense for 2-4hrs as lenses expand with heat Microscope also needs to be stable

Your microscope: temperature control Heat within the chamber holder Warm air stream over the stage Enclose the stage area Enclose the entire microscope

Your microsocope Active correction: – Autofocus-Not ideal: extra light exposure and change plane in x, y, z – Active Z position monitoring: Nikon and Zeiss Long term focus stability-important for time lapse work, not as important for short term observations with operator present

Perfect focus To overcome drift due to mechanical and thermal changes over time

Other features of microsocopes useful for cell imaging Keep the exposure constant Motorised stage to follow multiple cells (also need appropriate software) Shutter on illuminators so that the cells don’t bleach

Photodamage Live cells poorly tolerate high exposure to light- true for transillumination and epifluorescence: cell death, compromised cell function and stress Targets: the cell, the medium, the fluorophore Generation of reactive oxygen species Blue light is very toxic to cells The longer the wavelength, the better You have to compromise!

Light flux at specimen Illumination system: 75W Xenon arc 490/10nm exciter filter (60%T) 505nm dichromatic mirror (85% reflectance) Flux at specimen: 380W/cm times the flux of sunlight on the brightest day!

Minimize the exposure to the necessary for your experiment, not to make a pretty movie Kinetochore tracking in 3D 20 z-sections Every 7.5s seconds 5 minutes That’s a lot of exposure!

Minimum exposure to reduce photodamage Use a minimal exposure to maximize your data collection. Kinetochores are still there after 4min! Deconvolution (1cycle) can help restore your signal for presentation purposes.

Correcting for photobleaching

Type of live-cell imaging experiments one might do Time-lapse imaging (BF or TIRF) Photoactivated localized microscopy-PALM Fluorescence Recovery After Photobleaching-FRAP Fluorescence Correlation Spectroscopy-FCS Fluorescence Speckle Microscopy-FSM Fluorescence Resonance Energy Transfer-FRET

TIRF imaging of cells to image processes close to the membrane and focal adhesion

TIRF resolution in live-cell imaging nm in z-axis The evanescent field, resulting from total internal reflection of the beam excites fluorophores in a SMALL volume, close to the coverslip. Therefore sample photobleaching is very low

Fluorescence Recovery After Photobleaching- FRAP to look at 2D diffusion Very good for membrane dynamics

Photoactivation to determine movement of molecules and lifetime of subcellular structures

Fluorophore Photoconversion EosFP is a green fluorescent protein (emits at 516nm) from stony coral Near-UV radiation induces a conformational change in the protein Protein emission at 581nm Especially good for cell tracking in organisms

The birth of speckle microscopy

Fluorescence speckle microscopy to look at motion and turnover of macromoleulcar assemblies Courtesy of M. Mendosa/S. Besson

FSM gives information on flux and movement of actin during migration Courtesy of M. Mendosa/S. Besson

Quantitative analysis of FSM imaging gives information on actin movement during cell migration Courtesy of M. Mendosa/S. Besson

Fluorescence resonance energy transfer (FRET) FRET involves non-radiative energy transfer between donor and acceptor fluorophores Occurs over distances of 1-10 nm Emission and excitation spectrum must significantly overlap Can be used to measure close interaction between fluorophores and as a ‘spectroscopic ruler’ to measure intermolecular distance Donor molecule Acceptor molecule Excitation Emission Excitation Emission FRET Intensity Wavelength

Example: the emission and absorption spectra of cyan fluorescent protein (CFP, the donor) and yellow fluorescent protein (YFP, the acceptor), respectively. CFP & YFP pair is currently the ‘best’ for FP-based FRET. Fluorescence resonance energy transfer (FRET)

When to use FRET?

An Aurora B FRET probe as a tool to monitor differential phosphorylation FRET occurs when it is not phosphorylated Violin et al Fuller et al We;burn rt al, 2010 Donor Acceptor

Aurora B phosphorylation varies with substrate position Decreasing phosphorylation Michael Lampson, Dan Liu

Fluorescence resonance energy transfer (FRET) Intensity Wavelength Donor molecule Acceptor molecule Excitation Emission No FRET Intensity Wavelength Donor molecule Acceptor molecule Excitation Emission FRET An important control in FRET studies is to photobleach the acceptor and demonstrate that donor emission does NOT decrease